Using Biophysics to Understand the Basis of Allosteric Inhibition in the Hepatitis C Virus Polymerase

July 28 is World Hepatitis Day, drawing attention to the 240 million people chronically infected with the hepatitis B virus and 150 million people with the hepatitis C virus. To recognize this worldwide awareness day, BPS asked member Ian Thorpe, Assistant Professor of Chemistry and Biochemistry at the University of Maryland Baltimore County, to answer a few questions about his research on hepatitis C.

HCV blog

What is the connection between your research and hepatitis?

We study the RNA polymerase responsible for replicating the Hepatitis C virus (HCV) genome. There is no vaccine for HCV infection and this enzyme has been extensively targeted with the goal of developing HCV therapeutics. One reason for this is the crucial role the enzyme plays in generating new viral particles. In addition, because it is an RNA-dependent RNA polymerase, there is no homologous human version of this enzyme. Thus, if this enzyme is targeted there should be a decreased likelihood of generating side effects from impacting human proteins. The search for therapeutics has yielded many small molecule inhibitors of the polymerase, including allosteric inhibitors that bind distal to the active site. We employ molecular modeling and simulation to understand the physical processes that underlie this allosteric regulation.

Why is your research important to those concerned about these diseases?

Understanding how allosteric inhibitors modulate the function of the HCV polymerase provides insight into what intrinsic properties of the enzyme allow it to effectively replicate viral RNA. In addition, understanding the underlying molecular mechanisms involved may allow for the development of novel and more effective inhibitors. While there are treatments currently available to treat this disease, these are generally expensive, can require significant time investments and often have serious side effects. In addition, the HCV RNA polymerase does not contain proofreading ability. The resulting high error rate during replication induces an elevated incidence of mutations in the virus and facilitates the development of viral resistance to treatment regimens. Thus, there is a continuing need to identify new molecules that could serve as HCV therapeutics.

How did you get into this area of research?

During my postdoctoral position at the University of Utah with Professor Gregory Voth (now at the University of Chicago), I began to consider research projects that I could pursue during my independent career. My wife worked in the field of liver transplantation at the time. She was familiar with HCV because it is one of the leading reasons for liver transplantation in the United States. She also recognized the need for additional studies of HCV due to the limited treatment options and extensive gaps in our knowledge of how the virus functions. HCV infection is a burgeoning health crisis because the virus was only conclusively identified within the last thirty years and many people were likely infected (for example via blood transfusions) before a test became available to screen for HCV infection. Those who are infected often go decades without displaying symptoms, only to be diagnosed later with serious complications including cirrhosis and liver cancer. Thus, a large fraction of the population who were infected in the past may be asymptomatic and are only now starting to display symptoms (or will do so in the near future).

How long have you been working on it?

I began working in this field after becoming a faculty member in the Department of Chemistry and Biochemistry at the University of Maryland, Baltimore County in 2009.

Do you receive federal funding for this work? If so, from what agency?

I do not currently receive federal funding for this work. However, I do receive federal support in the form of compute time on supercomputing resources supplied by the National Science Foundation via the Extreme Science and Engineering Discovery Environment (XSEDE).

Have you had any surprise findings thus far?

Yes, several! One of the key features of the HCV polymerase is that it likely undergoes transitions to distinct conformational states as it replicates RNA. These include a closed state required for the initiation of replication and an open state associated with elongation of the newly synthesized RNA strand. We have discovered that components of the polymerase may have a regulatory function by restricting the conformational sampling of the enzyme. In addition, we have seen that while diverse allosteric inhibitors can induce distinct effects on conformational sampling and enzyme dynamics, shared characteristics exist that may underlie the inhibitory action of these small molecules. Specifically, most inhibitors we have studied disrupt the conformational sampling of the enzyme in ways that can be related to their inhibitory capability. Some discourage conformational transitions by overly stabilizing one or the other conformational state, while others may destabilize both states to the extent that neither can be stably occupied. Finally, we have observed that free enzyme is able to explore both the open and closed states thought to have functional roles in RNA replication. This result is unanticipated given that the free enzyme lacks other components of the replication complex such as RNA template or nucleotides. This observation suggests that the presence of ligands does not engender new enzyme conformational states, but instead shifts the populations of preexisting enzyme conformations. Such a phenomenon is consistent with the conformational selection model of allostery.

What is particularly interesting about the work from the perspective of other researchers?

Our studies highlight mechanisms of allosteric regulation that provide insight into how allosteric inhibitors decrease enzyme activity and suggest experiments that can be carried out to validate our hypotheses. Understanding the molecular mechanisms of allosteric inhibition is important in the development of HCV therapeutics because this knowledge may allow the discovery of new and more effective inhibitors or new ways of using existing inhibitors, such as in novel combination therapies. These studies also illuminate the fundamental processes involved in RNA replication by viral polymerases. HCV is related to several viruses that are serious human or agricultural pathogens, including the viruses that cause Dengue Fever, Yellow Fever, West Nile disease and Bovine viral diarrhea. Thus, knowledge gained from studying the HCV polymerase may be applicable to polymerases from these related viruses as well. More generally, our research helps to elucidate the many ways that allosteric regulation of enzyme function can occur. Allosteric regulation is a key way in which protein function can be modulated in biological systems. Thus, better comprehending the underpinnings of allosteric regulation may be applicable in diverse contexts such as in determining the molecular origin of disease states or in discovering drugs to treat other ailments.

What is particularly interesting about the work from the perspective of the public?

While this work is fundamentally basic science research, the knowledge we obtain may ultimately be useful in identifying novel and more effective treatment options for infection by HCV and related viruses.


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